64477024 1 Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide 1 Monooxygenase (LPMO) from Aspergillus fumigatus and investigation of its synergistic 2 effect using biomass. 3 Musaddique Hossain, Subba Reddy Dodda, Bishwajit Singh Kapoor, Kaustav Aikat, and 4 Sudit S. Mukhopadhyay* 5 Department of Biotechnology, National Institute of Technology Durgapur-713209, West 6 Bengal, India 7 Running title: Biochemical, structural insights, and investigation of the synergistic effect of 8 newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) from 9 Aspergillus fumigatus. 10 * To whom the corresponding author should be addressed. 11 E-mail: suditmukhopadhy@yahoo.com 12 Phone: +919434788139 13 14 15 16 17 18 19 20 21 22 23 24 25 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 2 Abstract 26 The efficient conversion of lignocellulosic biomass into fermentable sugar is a bottleneck for 27 the cheap production of bio-ethanol. The recently identified enzyme Lytic Polysaccharide 28 Monooxygenase (LPMO) family has brought new hope because of its boosting capabilities of 29 cellulose hydrolysis. In this report, we have identified and characterized a new class of 30 auxiliary (AA16) oxidative enzyme LPMO from the genome of a locally isolated 31 thermophilic fungus Aspergillus fumigatus (NITDGPKA3) and evaluated its boosting 32 capacity of biomass hydrolysis. The AfLPMO16 is an intronless gene and encodes the 29kDa 33 protein. While Sequence-wise, it is close to the C1 type of AaAA16 and cellulose-active 34 AA10 family of LPMOs, but the predicted three-dimensional structure shows the 35 resemblance with the AA11 family of LPMO (PDB Id: 4MAH). The gene was expressed 36 under an inducible promoter (AOX1) with C-terminal His tag in the Pichia pastoris. The 37 protein was purified using Ni-NTA affinity chromatography, and we studied the enzyme 38 kinetics with 2,6-dimethoxyphenol. We observed polysaccharides depolymerization activity 39 with Carboxymethyl cellulose (CMC) and Phosphoric acid swollen cellulose (PASC). 40 Moreover, the simultaneous use of cellulase cocktail (commercial) and AfLPMO16 enhances 41 lignocellulosic biomass hydrolysis by 2-fold, which is highest so far reported in the LPMO 42 family. 43 44 Importance 45 The auxiliary enzymes, such as LPMOs, have industrial importance. These enzymes are used 46 in cellulolytic enzyme cocktail due to their synergistic effect along with cellulases. In our 47 study, we have biochemically and functionally characterized the new AA16 family of LPMO 48 from Aspergillus fumigatus (NITDGPKA3). The biochemical characterization is the 49 fundamental scientific elucidation of the newly isolated enzyme. The functional 50 characterization, biomass degradation activity of AfLPMO16, and cellulase cocktail 51 (commercial) combination enhancing the activity by 2-fold. This enhancement is the highest 52 reported so far, which gives the enzyme AfLPMO16 enormous potential for industrial use. 53 54 Keywords: A.fumigatus, Auxiliary activity, Cloning, Kinetics, LPMO, Lignocelluloses, 55 Molecular docking 56 57 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 3 Introduction 58 The diminution of fossil fuels and the growing concern of environmental consequences, 59 particularly climate changes, have steered our fast-growing economy for clean and renewable 60 energy production [1]. Among different renewable energy sources, bioethanol is one of the 61 promising alternatives to fossil fuel because of its low CO2 emission [2, 3] and its 62 manufacturing reliance on lignocellulosic biomass, which is bio-renewable and abundance on 63 earth. However, the structural complexity and the recalcitrance of this renewable carbon 64 source [4] have hindered its optimal use. The current process of saccharification of 65 lignocellulosic biomass is time-consuming and costly. Therefore, the requirement of cost-66 effective and fast controlled destruction of lignocellulose has driven the bioethanol industry 67 to explore the accessory enzymes to achieve a better and efficient enzyme cocktail for the 68 commercial production of lignocellulose-derived ethanol. 69 A breakthrough in such exploration came into existence when a mono-copper redox enzyme, 70 known as Lytic polysaccharide monooxygenase (LPMO), was first reported in 2010 [5-8]. 71 LPMO increases lignocellulosic biomass conversion efficiency[9,10 ] by catalyzing the 72 hydroxylation of C1 and/or C4 carbon involved in glycosidic bonds that connect glucose unit 73 in cellulose and allow cellulase enzymes to process the destabilized complex polysaccharides 74 [11-15]. Harris et al., in their study, used LPMO from T reesei along with classical cellulases 75 and showed that the degradation of polysaccharide substrates was increased by a factor of 76 two when compared with the activity of classical cellulases alone [16]. A CBM33 domain-77 containing enzyme identified from Serratia marcescens with boosting chitinase activity, later 78 classified as LPMO. A study by Nakagawa et al. showed that an AA10 family of LPMO from 79 Streptomyces griseus could increase the efficiency of chitinase enzymes by 30- and 20-fold 80 on both α and β forms of chitin, respectively [17]. Along with this work, there are some 81 recent reports of the synergistic effect of LPMOs with glycoside hydrolases on 82 polysaccharide substrates [18-20]. 83 LPMOs are classified as AA9, AA10, AA11, AA13, AA14, and AA15 in the CAZy database 84 (http://www.cazy.org/), based on their amino acid sequence similarity. Recently Filiatrault-85 Chastel et al. identified the AA16, a new family of LPMO from the secretome of a fungi 86 Aspergillus aculeatus (AaAA16). The AaAA16 was initially isolated as X273 protein 87 (unnamed domain) and later identified as C1-oxidizing LPMO active on cellulose [21]. 88 AaAA16, the only AA16 family of LPMO so far, has been identified, and it lacks complete 89 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 4 biochemical characterization. The biochemical characterization, structural characterization, 90 and the assessment of biomass conversion efficiency are required to understand better the 91 action of members of this new family on plant biomass and their possible biological roles. 92 While we were analyzing the cellulose hydrolyzing genes from the genome of A. fumigatus 93 (Aspergillus genome database), we identified five LPMOs, one belonging to AA16 family 94 because of its X273 domain. Further, we cloned the AfLPMO16 gene from the genome of our 95 locally isolated strain of A. fumigatus (NITDGPKA3) [22] (GenBank accession No. 96 JQ046374) by designing the primers based on the A. fumigatus LPMO sequence 97 (CAF32158.1)(NCBI). The cloned A. fumigatus (NITDGPKA3) LPMO (after cloning and 98 sequencing the sequence submitted to GenBank; accession No. MT462230) is expressed in 99 Pichia pastoris X33. The heterologous protein (AfLPMO16) purified and used for 100 biochemical and functional characterization. The saccharification rate assessment suggests 101 that AfLPMO16 has fast and effective glucose releasing ability from lignocellulose and 102 cellulose when used with a commercial cellulase cocktail. Enzyme kinetics using 2,6-103 dimethoxyphenol as a substrate [23] confirmed the oxidative activity. The lignocellulosic 104 biomass (alkaline pre-treated raw rice straw) conversion efficiency along with cellulases 105 suggests that AfLPMO16 could be an essential member of the cellulase cocktail for industrial 106 use. 107 Results 108 Cloning, expression, and purification of AfLPMO16 109 AfLPMO16 (GenBank accession No. MT462230) is an intronless 870 nucleotide long gene 110 that encodes 290 amino acids. The theoretical molecular mass is 29KDa (including signal 111 peptide). The gene sequence of AA16 from our isolated strain of A.fumigatus (NITDGPKA3) 112 has shown almost 99.6% homology with the gene sequence of AA16 present in the genome 113 database of A.fumigatus (CAF32158.1) (NCBI database). 114 The protein of AfLPMO16 (GenBank accession No. MT462230) was produced in Pichia 115 pastoris X33 without its C-terminal extension. After the optimization of the expression 116 procedure, we achieved approximately 0.8 mg/ml of purified protein. The SDS-PAGE 117 analysis (Fig 1) confirmed the single band of the purified protein (Fig. 1: lanes 5 and 6). We 118 further confirmed the purified recombinant protein bearing the 6X His-tag by Western blot 119 using an anti-His antibody (Fig. 1: Lane W1 & W2); the purified protein (lane 5 & 6 of SDS-120 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 5 PAGE) used for western blot. The expressed recombinant AfLPMO16 band appeared at 121 approximately 32kDa position in SDS-PAGE (Fig. 1), which is slightly higher than the 122 expected size. It is probably due to glycosylation [24], or recombinant protein has c-myc 123 epitope and 6x His tag in its c-terminal that can increase the molecular mass by 2.7KDa. For 124 further confirmation of N-glycosylation, we checked the AfLPMO16 sequence glycosylation 125 site using NetNGlyc 1.0 server (DTU Bioinformatics, Technical University of Denmark, 126 http://www.cbs.dtu.dk/services/NetNGlyc/) [36]. There were two N-glycosylation sites 127 present above the 0.5 threshold value at 114 & 149 amino acid sequence positions with 0.76 128 and 0.56 potential values, respectively. 129 Enzyme assay and Kinetics 130 LPMO converts 2,6-dimethoxyphenol (2,6-DMP) into 1-Coerulignone (Fig. 2a) due to its 131 oxidative property, and 1-Coerulignone has an extinction coefficient of 53200 . 1-132 Coerulignone gives absorbance at 469nm wavelength; therefore, we can easily quantify it 133 using a spectrophotometer [21]. The OD at 469nm wavelength steadily increases with time 134 that clearly indicates the steady conversion of 2,6-dimethoxyphenol to 1-Coerulignone (Fig. 135 2a). It also suggests the sufficient activity of the enzyme AfLPMO16. Temperature and pH 136 influence the activity of LPMO. Thus, during the kinetic study, we used optimum 137 temperature 30 and pH 6.0, as described by [21]. AfLPMO16 showed proper activity for 138 the chemical substrate 2,6-dimethoxyphenol; there was a steady release of 1-Coerulignone 139 when incubated 2,6-dimethoxyphenol with AfLPMO16. The enzyme kinetics was performed 140 with different concentrations of 2,6-dimethoxyphenol. We obtained the Kinetics parameters 141 such as Michaelis Menten constant (Km) and maximum velocity (Vmax) from the Line-142 weaver-Burk plot (Fig. 2b) as 5.4mM, and 0.153 U/mg, respectively. The calculated catalytic 143 activity Kcat was 277.67 min -1 (Table 1). These kinetics parameters suggest that the oxidative 144 property of AfLPMO16. 145 In-silico analysis for substrate specificity 146 The AfLPMO16 contains 19 amino acids long N-terminal signal peptide before His1 catalytic 147 domain (1-169aa), and C terminal Serine rich region (170-271aa) (Fig. 3a). This N-terminal 148 sequence is one of the marker features of fungal LPMOs, but this serine-rich C-terminal or 149 linker is a feature of AA16 family. It also lacks the CBM1 module or 150 glycosylphosphatidylinositol (GPI) anchor, like other AA16 LPMOs [19]. AfLPMO16 also 151 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 6 has conserved Histidine at 1st and 109th positions, which are mainly involved in copper 152 binding, the signature characteristic of LPMOs. There are other conserved sequences like 153 Gly, Pro, Asn, Cys, Try, Tyr, Leu, and Asp, including GNV(I)QGELQ motif (Fig. 3b) The 154 fully conserved sequences (highlighted with red background) are the marker amino acids 155 represent the LPMOs. The partially conserved sequences (within the blue boxes) are the 156 marker of different auxiliary families (Fig. 3b). The sequence alignment studies of AA16 157 family (including AfLPMO16) with other families (AA9, AA10, and AA11) of LPMOs 158 suggested (Fig. S1) a co-relation between AA10 family and AA16 LPMOs. The substrate-159 binding motif in the L2 loop of cellulose active LPMO10 has some similarities with AA16 160 L2 loop motif (marked with black box) and cellulose active motif (Fig. 3b). In AA16 LPMOs 161 the conserved motif in L2 loop GNI(V)QGEL the region is replaced by YNWFG(A)NL for 162 C1 oxidizing AA10 LPMOs, which are also cellulose active. The previous study suggests that 163 the amino acids (Y79, N80, F82, Y111, and W141) in loop L2 take part in substrate 164 specificity for LPMO 10, and mutations (Y79, N80D, F82A, Y111F, W141Q) alter the 165 specificity of the substrate from chitin to cellulose [37]. In AfLPMO16, the corresponding 166 amino acids GNQYR (Fig. 3b) (marked with black arrows), some amino acids from these 167 positions (N & Y) are also present in cellulose-active AA10 LPMOs. Hopefully, the polar 168 amino acids (Q & R) are charged and may interact with chitin due to electrostatic interaction. 169 Alternatively, there are high chances that few mutations in these amino acids may help 170 AfLPMO16 to interact with chitin. Further, in chitin active LPMOs, more than 70% residues 171 of the motif (Y(W)EPQSVE) are polar, including two negatively charged Glu (E). In 172 cellulose active LPMOs, 70% residues of the motif (Y(W)NWFGVL) are hydrophobic [38]. 173 In contrast, in AfLPMO16, 70% residues are polar, including one negatively charged Glu (E), 174 one hydrophobic Tyr (Y), and others are neutral. The presence of polar residue and negative 175 charged Glu (E) suggests that AfLPMO16 may bind to chitin. Electrostatics interaction 176 between the substrate and enzyme active site plays a pivotal role in substrate binding. The 177 electrostatic potential surface at the catalytic site of the AfLPMO16 was found unchanged or 178 slightly positive-charged at pH 6.0 (Fig. 3c) (Marked in the figure). The electrostatic 179 interaction study suggests that the AfLPMO16 may also bind to cellulose [52]. 180 Regioselectivity of AfLPMO16 181 Amino acids on the substrate-binding surface determine the oxidative regioselectivity of 182 LPMOs [29]. Sequence comparison and mutation studies revealed that the conserved amino 183 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 7 acids near the catalytic center in C1 and C1/C4 oxidizing AA10 and AA9 LPMOs are 184 responsible for regioselectivity. In the case of C1/C4 oxidizing AA10, the amino acid Asn85 185 near the catalytic center is responsible for C4 oxidizing activity. Alteration of this amino acid 186 (N85F) diminished the C4 activity and produced only C1 oxidized product [39]. In C1 187 oxidizing AA9 LPMOs, hydrophobic amino acids Phe and Tyr are conserved in addition to 188 Asn. While in C1 oxidizing AA10 LPMOs, the Phe amino acid has replaced the 189 corresponding Asn site (Fig. 3b)(marked with red arrow). The Phe is also parallel to the 190 substrate-binding surface [47]. In AA16, the corresponding Gln (Q) may be parallel to the 191 substrate-binding region (Fig. 3b). The function of conserved Gln (Q) is not clear. However, 192 this polar amino acid has a similar side chain with polar Asn (N). The axial distance between 193 the conserved amino acid and copper catalytic center is another crucial factor for 194 regioselectivity. The C1/C4 oxidizing AA10 LPMOs have more open or wider axial gaps 195 than C1 oxidizing AA10 LPMOs [39]. Here the distance between Gln56 and His20 is 7.7Å, 196 and the distance between Gln56 and Cu catalytic center is 11.1Å. In the absence of the AA16 197 structure (crystal or model), we cannot compare the lengths; nevertheless, this distance may 198 play a key role in regioselectivity. 199 Phylogenetic tree construction and analysis 200 The sequential and functional relationship of AA10 and AA16 LPMOs has been discussed, 201 but phylogenetic studies based on the sequence similarity give an evolutionary origin. Based 202 on sequence comparison, AfLPMO16 is evolutionarily closer to the LPMO of Aspergillus 203 fisheri (91% sequence homology). The constructed phylogenetic tree contains two main 204 clades and two subclades (Fig. 4). The first clade contains all AA10 LPMOs from bacterial 205 species such as Bacillus thuringiensis, Bacillus amyloliquefaciens, Streptomyces lividans, and 206 Enterococcus faecalis. The second clade includes all fungal AA10 and AA16LPMOs, mainly 207 belongs to Aspergillus, and Penicillium species in which AA16 LPMOs are mostly from 208 A.niger, A.fumigatus, A.fisheri, Aspergillus kawachii (Fig. 4). 209 Model structure prediction and molecular docking analysis 210 I-TASSER was used to predict the three-dimensional structure of the AfLPMO16. Most of the 211 LPMOs have immunoglobulin-like distorted β-sandwich fold like structures, in which loops 212 connect seven antiparallel β-strands with a different number of α-helix insertions (Fig. 5a). 213 The final model has a β-sandwich structure connected by loops with two α-helices. The 214 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 8 superimposition of the AfLPMO16 with other LPMO families like AA9, AA10, AA11, and 215 AA13 showed that they share common antiparallel β-strands and helices with more loops, 216 which indicate higher flexibility. Moreover, AfLPMO16 showed 1.2Å RMSD with AA11 217 (PDB Id: 4MAH) LPMO lower than the other LPMOs. So the 3D structure of AfLPMO16 218 suggests that it has more structural resemblance with AA11 LPMO. We also found one 219 disulfide bond in AfLPMO16 between the Cys78-Cys186 amino acids, signature of thermo-220 stability (Fig. S2). The histidine brace amino acids, such as His20 and His109, participate in 221 coordination bond with Cu ions. The surface of AfLPMO16 has an active site (Fig. 5b). The 222 interaction studies with cellohexose suggest amino acids like Gln48, Gln181, Ser178, His109, 223 His20, Asn54, Asp50, Tyr52, and Glu58 (Active enzyme starts with His1; so His20 will His1 224 and corresponding amino acids can be numbered accordingly) are in the active site and are 225 involved in the interaction with the substrate (Fig. 5c). Molecular docking suggests that 226 AfLPMO16 has a cellulose-binding surface (Fig. 5b & 5c). This study also suggests that the 227 binding energy between AfLPMO16 and cellulose is -7.0 kcal/mol, which is highest 228 compared to chitin (-5.5kcal/mol) and other polysaccharides. 229 Polysaccharides depolymerization by AfLPMO16 230 AfLPMO16 showed efficient depolymerization activity on both CMC and PASC (Fig. 6a & 231 6b). We quantified the amount of reducing sugar released by enzymatic degradation. When 232 incubated CMC with increasing concentrations of the enzyme, the amount of product 233 (reducing sugar) increased with the increase of AfLPMO16 concentration (Fig. 6a). When we 234 added 50µg of the enzyme, nearly 0.05mg/ml of reducing sugar was released. For 100µg of 235 the enzyme, the product was nearly 0.136mg/ml, and for 200µg of the enzyme, the amount of 236 product released was approximately 0.356mg/ml (Fig. 6a). This result indicates the 237 polysaccharide (CMC) depolymerization activity of AfLPMO16. 238 Further, we used insoluble PASC as a substrate and incubated with an increasing 239 concentration of AfLPMO16, and determined the relative absorbance of PASC with the 240 growing amount of enzyme. The enzyme degrades the polysaccharide (substrate) into smaller 241 polysaccharide units (monosaccharides, disaccharides, etc.), which are soluble and make the 242 reaction mixture clearer. Therefore, it leads to a decrease in the absorbance resulting 243 increment in relative absorbance [40]. Ultimately we will find a graph where relative 244 absorbance increase with increasing concentration of AfLPMO16. Hence In this experiment, 245 we found a rise in relative absorbance concerning the untreated substrate with a high 246 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 9 concentration of enzyme AfLPMO16 (Fig. 6b). The graph (Fig. 6b) showed that 0.17 247 absorbance difference concerning untreated substrate when we used 50µl (concentration 248 0.8µg/µl) of the enzyme. The difference in absorbance steadily increased with the escalation 249 of enzyme concentration (200µl of the enzyme at the concentration of 0.8µg/µl the relative 250 absorbance reached nearly 0.36). Hence these experiments confirmed the intrinsic 251 polysaccharide degradation property of the AfLPMO16 like other LPMOs. In these 252 experiments, we used the heat-inactivated AfLPMO16 and ascorbic acid-deficient set to 253 verify these results (data not shown). 254 Pre-treated lignocellulosic biomass and cellulose hydrolysis with simultaneous treatment of 255 AfLPMO16 and commercial cellulase 256 There are two modes of action to show the synergy or boosting effect of LPMO while using 257 with cellulase- sequential assay and simultaneous assay. In the sequential assay, LPMO 258 should add a prior time limit to cellulase. And in the simultaneous assay, both the enzymes 259 LPMO and cellulase are being used together to the substrate. In this study, we chose to 260 perform a simultaneous assay for two reasons; simultaneous assay shows better synergy or 261 boosting in crystalline cellulose [41] than sequential one. Furthermore, we aimed to check the 262 synergy or stimulating activity of commercial cellulase by AfLPMO16 so that it may include 263 in the cocktail for better depolymerizing action. Here the boosting effect of AfLPMO16 was 264 studied with a commercial cellulase cocktail on both cellulose (Avicel) and lignocellulosic 265 biomass (alkaline pre-treated rice straw). The alkaline pre-treatment has a beneficiary over 266 acid pre-treatment in terms of hydrolysis yield [48]. The reason is that alkaline pre-treatment 267 sufficiently removes the lignin [42], but it preserves hemicelluloses [43]. When incubating 268 Avicel with AfLPMO16 and cellulase, the amount of reducing sugar released was almost 269 double compared to Avicel incubated with either cellulase alone or cellulase along with heat-270 inactivated AfLPMO16 (Fig. 7b). A similar kind of boosting effect we observed in every 271 time point from 5 hrs to 72 hrs. We also found the synergistic impact of AfLPMO16 in 272 lignocellulosic biomass transformation to fermentable sugar (Fig. 7a). When we incubated 273 the alkaline pre-treated rice straw with 100 µg and 200µg of AfLPMO16 along with cellulase, 274 almost 1.7 fold and slightly above 2-fold of reducing sugar were released respectively 275 compared to lignocellulose incubated with either cellulase alone or cellulase along with heat-276 inactivated AfLPMO16 (Fig. 7a) suggests the enhancement is dependent on the amount of 277 auxiliary enzyme AfLPMO16. For further elaboration of the synergistic effect of AfLPMO16, 278 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 10 another set of reactions prepared where the biomass was treated with an increasing 279 concentration of only AfLPMO16. A minimal amount of hydrolysis activity was there, nearly 280 0.04 mg/ml to 0.06 mg/ml, reducing sugar quantified for AfLPMO16 treated biomass (Fig. 281 7c). This hydrolysis activity of AfLPMO16 alone is negligible compare to only cellulase 282 treated biomass. 283 Nevertheless, the simultaneous use of AfLPMO16 and cellulase enhances the hydrolysis 284 activity two-fold compared to the only cellulase treated biomass (Fig. 7c). This result 285 strongly indicates the synergistic effect of AfLPMO16 with cellulase. All these results 286 confirmed the boosting effect or synergistic effect of AfLPMO16 on the hydrolytic activity of 287 cellulase for both cellulosic and lignocellulosic biomass degradation. So far highest 288 synergistic effect was reported by AA9 (Table 2), which is less than two-fold [44, 45]. 289 Discussion 290 The gene was cloned in pPICZαA vector under the control of AOX1 promoter by following 291 the same strategy developed for AaAA16 and PMO9A_MLACI [19, 26]. The nucleotide 292 sequence of AfLPMO16 was codon-optimized for Pichia pastoris. The recombinant protein 293 containing a C-terminal polyhistidine tag was produced in flasks in the presence of trace 294 metals, including copper, and purified from the culture supernatant by immobilized metal ion 295 affinity chromatography (IMAC: Ni-NTA affinity chromatography), following the same 296 protocol used for AaAA16 [19]. We were successful in producing the active AfLPMO16 in 297 P.pastoris X33 (Fig. 1) in a shake flask. Despite the chance of N-terminal modification in 298 shake flask culture instead of bioreactor culture [19], the amount of active enzyme obtained 299 in shake flask was sufficient for characterization. The enzyme activity determined by 2,6-300 dimethoxyphenol concerning the heat-inactivated enzyme and without ascorbic acid as 301 negative controls (data not shown). The enzyme activity suggests the successful production 302 of active protein (Fig. 2a), and interestingly, the initial reaction rate is faster compared to later 303 time span. Lytic polysaccharide monooxygenase (LPMO) releases a spectrum of cleavage 304 products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct 305 identification and quantitation of these released products is the basis of MS/HPLC-based 306 detection methods for LPMO activity, which is time taking and is required specialized 307 laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay 308 based on the 2,6-dimethoxyphenol can accurately measure the enzymatic action and can be 309 used for enzyme screening, production, and purification, and can also be applied to study 310 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 11 enzyme Kinetics [21]. Thus it is swift, robust for biochemical characterization, and also 311 accurately determines the active enzyme. 312 Sequence analyses indicating that the AfLPMO16 has some signature characteristics for both 313 cellulose and chitin-binding and both C1 and C1/C4 oxidizing activity. However, 314 experimental confirmation is required to establish the presence or absence of any chitin-315 binding nature and C1/C4 oxidizing capability of AfLPMO16. The constructed phylogenetic 316 tree (Fig. 4) suggests that the fungal AA10 and AA16 LPMOs are more likely to come from a 317 common ancestor. Molecular docking study suggests that AfLPMO16 has the highest affinity 318 towards cellulose among the known substrates, based on the binding energy. The binding 319 energy between cellulose and AfLPMO16 is -7.0 Kcal/mol, which makes thermodynamically 320 strong binding between enzyme and substrate (Fig. 5b & 5c) compared to other substrates. 321 The LPMOs are essential for their auxiliary activity and polysaccharide degrading property. 322 We observed polysaccharide depolymerizing activity on carboxymethyl cellulose (CMC) and 323 phosphoric acid swollen cellulose (PASC) (Fig. 6a & 6b). Due to its auxiliary activity, it 324 enhances the action of the cellulase enzyme for the degradation of cellulose and 325 lignocelluloses [49]. The only identified AA16 family, the AaAA16, showed a sequential 326 boosting effect with T. reesei CBHI on nano-fibrillated cellulose (NFC) and PASC. The 327 AaAA16, the recent addition of the AA16 family of LPMO in the CAZY database, showed 328 synergism with the CBH1 for the degradation of cellulose [19]. However, AaAA16 study did 329 not deal with the biomass hydrolysis boosting effect of the AA16 family. The boosting result 330 is most important in the technical aspect for enhancing the activity of the cellulase cocktail. 331 LPMO enzyme has earned much research interest due to their synergistic effect or boosting 332 effect on cellulase enzyme [45]. AfLPMO16 showed a boosting impact on cellulose and 333 lignocellulose hydrolysis (Fig. 7a & 7b). The synergism of AfLPMO16 has shown in (Fig. 334 7c), where the only AfLPMO16 and only cellulase treated biomass hydrolysis activity is low 335 compare to the combined effect of these two enzymes. The simultaneous use of AfLPMO16 336 and cellulase enhances nearly two-fold biomass hydrolysis compare to the only cellulase 337 treated biomass hydrolysis. This enhancement of two-fold biomass hydrolysis is higher than 338 that of other LPMO families [50]. However, the synergy or boosting effect depends on many 339 factors such as pre-treatment [51], the lignin content of lignocelluloses and acting cellulase 340 [46]. Still, over 50% enhancement suggests intense demands on inclusion on cellulase 341 cocktail. However, the mechanism of synergism with the cellulase enzyme complex is poorly 342 understood. The probable explanation of such a boosting effect could be that the cellulosic 343 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 12 biomass is partially depolymerized by the LPMO, which gives further access to the cellulase 344 enzymes. 345 Conclusion 346 In concluding remark, AfLPMO16 is the second report of the AA16 family of LPMO, but for 347 the first time, we have characterized the AA16 family biochemically and structurally. In-348 silico sequence analysis, structure analysis, and molecular docking studies suggest some 349 unique characteristics of the AfLPMO16, like cellulose-binding ability, chances of chitin-350 binding, and C1 and C4 oxidizing property. Further studies, including the engineering 351 approach, are required to confirm these characteristics. Nevertheless, the most crucial aspect 352 of AfLPMO16 is the significant boosting effect on commercial cellulase cocktail in 353 lignocellulosic biomass conversion, and that suggests its importance in the bioethanol 354 industry. 355 Materials and Methods 356 Sequence analysis and Phylogenetic analysis: 357 AfLPMO16 sequence (CAF32158.1) was obtained from NCBI, and the sequence was further 358 confirmed from the Aspergillus genome database (http://www.aspgd.org/). To avoid 359 interference from the presence or the absence of additional residues or domains, the signal 360 peptides, and C-terminal extensions were removed before the alignment. Homology sequence 361 alignment was performed by the BLAST [22]. Clustal Omega [23] was used for multiple 362 sequence alignment. The sequence alignment was edited with Espript for better visualization. 363 Pymol [24] and MEGA7 [25] were used to construct a phylogenetic tree after sequence 364 alignment. To build the phylogenetic tree, the sequences of twenty-seven (27) LPMO genes 365 (edited to remove N-terminal signal sequence, C-terminal extension or GPI anchor, CBM1 366 module) were taken from different species belong to AA10 and AA16 family of LPMOs. The 367 neighbor-joining tree was constructed with 1000 bootstrap replications. 368 Cloning of AfLPMO16 369 Aspergillus fumigatus NITDGPKA3 was grown on CMC agar media containing 2% CMC, 370 0.2% peptone, 2% agar in basal medium (0.2% NaNO3, 0.05%KCl, 0.05%MgSO4, 371 0.001%FeSO4, 0.1%K2HPO4). The fungal biomass was then milled in a pestle and mortar 372 followed by rapid overtaxing in solution with an appropriate lysis buffer for proper lysis of 373 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 13 the cell. Genomic DNA was isolated from the fungal biomass using the DNA extraction 374 buffer (400mM Tris-HCl, 150mM NaCl, 0.5M EDTA, 1%SDS) and followed by Phenol, 375 chloroform and isoamyl alcohol (25:24:1) extraction. The final pellet was washed with 70% 376 alcohol, air-dried, and dissolved in sterile water. AfLPMO16 gene was amplified by 377 polymerase chain reaction (PCR). The codon-optimized gene for Pichia pastoris was inserted 378 into the pPICZαA vector (Invitrogen Carlsbad, California, USA). The gene was cloned with 379 the native signal sequence and 6x His-tag at the C-terminal [26]. The cloning was done by 380 following the same protocol as AaAA16 and PMO9A_MLACI [19, 26]. The vector 381 (pPICZαA) containing the AfLPMO16 gene was linearized by Pme1 (New England BioLabs) 382 and transformed to Pichia pastoris X33 competent cells. The Zeocin resistant transformants 383 were picked and screened for protein production. The cloned gene was further confirmed by 384 sequencing and the sequence submitted to GenBank (GenBank accession No. MT462230). 385 Expression and purification of AfLPMO16 386 The positive colonies were selected on YPDS (Zeocin: 100μg/ml) plates. The positive 387 transformants were further screened by the colony PCR and expression studies. Protein 388 expression was carried out initially in BMGY media containing 1ml/L Pichia trace minerals 389 4 (PTM4) salt (2g/L CuSO4·5H2O, 3g/L MnSO4·H2 O, 0.2g/L Na2MoO4·2H2O, 0.02g/L 390 H3BO3, 0.5g/L CaSO4·2H2O, 0.5g/L CoCl2, 12.5g/L ZnSO4·7H2O, 22g/L FeSO4·7H2O, NaI 391 0.08g/L, H2SO4 1mL/L) and 0.1 g/L of biotin. Then after 16 hours, Pichia cells were 392 transferred into BMMY medium (PTM4 salt) with continuous induction by the addition of 393 1% methanol (optimized) every day (after every 24 hours) for three days. After three days, 394 the culture media was spun down (8,000rpm for 10mins) at 40C. The pellet was discarded, 395 and the media was collected. The protein was precipitated from the media by ammonium 396 sulfate precipitation (90% saturation). The pellet was redissolved in Tris buffer (Tris-HCl 397 50mM pH-7.8, NaCl-400mM, Imidazole-10mM). The recombinant protein was purified by 398 immobilized ion affinity chromatography (Ni-NTA affinity chromatography)[27], followed 399 by dialysis with 50mM phosphate buffer, pH 6.0. We followed the expression and 400 purification procedure, same as AaAA16 [19]. The yield of the purified protein was almost 401 0.8 mg/ml. The concentration was measured by Bradford assay, and BSA was used for 402 standard concentration. The protein was separated by SDS-PAGE using 12% acrylamide in 403 resolving gel(dH2O-3.6 ml, Acrylamide+Bisacrylamide – 4.0 ml, 1.5M Tris-2.6 ml, 404 10%SDS-0.1 ml, 10% APS-0.1 ml, TEMED- 0.01 ml; for 10 ml), stained with coomassie 405 blue, and the purified protein band was also confirmed by Western blot analysis by using an 406 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 14 anti-His antibody (Abcam). 407 Biochemical assays of AfLPMO16 408 Biochemical characterization of AfLPMO16 409 2,6 DMP (2,6-dimethoxyphenol) was used as a substrate for AfLPMO16 in this study. The 410 reaction was done in phosphate buffer (100mM pH 6.0) containing 10mM 2,6-411 dimethoxyphenol, 5μM hydrogen peroxide, and 50μg of purified AfLPMO16 at 30�C. The 412 amount of product 1-coerulignone was measured by spectrophotometer using the standard 413 extinction coefficient (53200M-1cm-1) and Lambert-Beer law. For kinetic assay different 2,6-414 dimethoxyphenol concentrations (1mM, 5mM, 10mM, 20mM, 25mM, 30mM, 40mM, 415 50mM, 70mM and 100mM) were used. The kinetic parameters were calculated based on the 416 Line-weaver-Burk plot (LB plot). One unit of enzyme activity is defined as the amount of 417 enzyme which releases 1μM of 1-coerulignone (product) per minute in standard reaction 418 condition. 419 Polysaccharides depolymerization by AfLPMO16 420 Different cellulosic compounds such as PASC, avicel®PH-101 (SIGMA), and carboxyl 421 methylcellulose (CMC) was used. We used 1% Avicel®PH-101 (SIGMA) (crystalline 422 cellulose) and 1% CMC (Carboxyl methylcellulose sodium salt) with different concentrations 423 of purified AfLPMO16 for different incubation time. Reducing sugar was determined by 424 Dinitro salicylic acid (DNS) assay. For PASC assay, we used 0.25% PASC and incubated 425 with increasing concentration of AfLPMO16 for 6 hours and measured the OD after 6hrs of 426 incubation and plot the relative absorbance ([OD of AfLPMO16 treated PASC]-[OD of 427 untreated substrate]) with enzyme concentration [28]. 428 Biomass and cellulose hydrolysis by cellulase and AfLPMO16 429 Cellulose and lignocellulose (alkaline pre-treated raw rice straw) [29] was used to determine 430 the cellulose hydrolysis enhancing capacity. Rice straw was pre-treated with 5% NaOH (1:10 431 W/V ratio) at 120�C at 15Psi pressure for 1 hour, and sodium azide (20%) 10μl (per 10ml) 432 was added at the reaction mixture to prevent any microbial contamination. The reaction was 433 performed at 50�C, and the amount of reducing sugar was quantified after 5hours, 24hours, 434 48hours, and 72 hours by Dinitro salicylic acid (DNS) assay. 20μl of cellulase (commercial) 435 (MP Biomedicals LLC) (5mg/ml) was used along with two different concentrations of 436 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 15 AfLPMO16 125μl (100μg) and 250μl (200μg) [concentration 0.8mg/ml]. Reaction sets were 437 prepared using the only cellulase, only AfLPMO16 with different concentrations, combined 438 AfLPMO16 and cellulase and lastly, cellulase with inactivated AfLPMO16. AfLPMO16 was 439 heat-inactivated by keeping at 100�C temperature for 30 minutes. Reducing sugar from each 440 triplicate sets were quantified. In the case of cellulose degradation, 400μl (1%) of avicel 441 (SIGMA) was incubated with 10μl of cellulase (commercial) (MP Biomedicals LLC) 442 (5mg/ml). Reducing sugar was quantified after 5 hours of incubation. For these biochemical 443 assays, we used 100mM phosphate buffer (pH-6.0), and heat-inactivated AfLPMO16 was 444 taken as a negative control. 445 Molecular modeling and Molecular docking 446 I-TASSER [30] server was used to model the AfLPMO16. The final model was energy 447 minimized by Gromacs software [31]. The Ramachandran plot [32] and Procheck [33] was 448 used to evaluate the final model. For Metal Ion-Binding site prediction and docking server or 449 MIB server (http://bioinfo.cmu.edu.tw/MIB/) were used to identify the copper (Cu) ion 450 position. A molecular docking study was performed by the Autodock Vina [34] using MGL 451 tools (Molecular graphics laboratory). The optimized substrate structures were prepared by 452 Autodock vina and saved in PDBQT format. The grid size parameters used in this docking 453 were 44, 46, 46, and grid center parameters used in this study were 49, 45, and 55. The 454 genetic algorithm was also used for docking. Molecular interactions between enzyme and 455 substrate were analyzed by the MGL tools [35]. The electrostatic potential surface of the 456 AfLPMO16 is calculated by the APBS plugin available in Pymol at pH 6.0. 457 458 Acknowledgments 459 MH is thankful to DBT, and SRD is grateful to DST Inspire for their fellowship. The authors 460 are also thankful to DST-FIST grant of the Department of Biotechnology, NIT Durgapur. 461 Funding 462 This study is financially supported by the DBT, Govt. of India (Grant No. BT/PR13127/ 463 PBD/26/447/2015). 464 Authors’ contribution 465 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 16 MH and SM designed the research work. MH, BSK, and SM wrote the manuscript. MH 466 performed biochemical assays. SRD performed In-Silico analysis. MH and KA analyzed the 467 results. All authors read and approved the manuscript. 468 Conflict of interest 469 Authors have no competing interests. The manuscript has been spell-checked, grammar 470 checked and plagiarism-checked by “Grammarly.” 471 Ethical approval 472 No human participants or animal is being used during the study. 473 474 References 475 1. Dias De Oliveira Me, Vaughan Be, Rykiel EJ (2005) Ethanol as Fuel: Energy, Carbon 476 Dioxide Balances, and Ecological Footprint. Bioscience. https://doi.org/10.1641/0006-477 3568(2005)055[0593:eafecd]2.0.co;2 478 2. 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BMC 632 Biochem 9:1–11. https://doi.org/10.1186/1471-2091-9-2 633 634 635 636 Figure legends 637 Figure 1 Expression and purification of AfLPMO16 (marked with red arrow). SDS PAGE 638 analysis; lane1, flow-through, lane2,3&4 wash, lane 5 & 6. Purified AfLPMO16: Western 639 blot analysis using purified protein presented in lane 5 & 6 of SDS page marked as lane W1 640 and W2 641 Figure 2 Enzyme kinetics studies of AfLPMO16 with 2,6-DMP (mean values are plotted). (a) 642 Chemical reaction to convert 2,6DMP to 1-coerulignone; OD at 469 nm vs. time plot. (b) LB 643 plot or 1/v vs 1/[s] plot. 644 Figure 3 In silico analysis of AfLPMO16. (a) Schematic diagram of AfLPMO16; signal 645 peptide: 19 amino acids, catalytic domain: 1-169 amino acids, and a serine-rich domain: 169-646 271 amino acids. (b) Multiple sequence alignment of AA16 LPMOs, C1 oxidizing, and 647 C1/C4 oxidizing AA10 LPMOs: Conserved sequences are highlighted. The red arrow 648 indicates the amino acid responsible for regioselectivity; the Black arrow represents the 649 amino acid responsible for substrate specificity, the black box represents the AA16 conserved 650 motif. (c) The electrostatic surface potential of AfLPMO16 model structure at pH6.0, blue 651 and red color represents positive and negative potential surface respectively. The area 652 surrounded by the ring represents the catalytic site. 653 Figure 4 Phylogenetic relationship of AfLPMO16 with AA10 LPMOs. A neighbor-joining 654 tree from MEGA showing C1(Bacterial) & C2(Fungal) clades and C2 clade further divided 655 into C2.1 ( Penicillium & other ) & C2.2 (Aspergillus) subclades. 656 Figure 5 Model structure and molecular docking of AfLPMO16. (a) Predicted three-657 dimensional models of the AfLPMO16 showing functional loops LS(orange), L2(blue), 658 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 22 L3(green), LC(magenta) loops surrounding the copper active site. (b) Histidine brace (His20, 659 His109) of AfLPMO16 surrounding the copper metal. (c) Amino acids involved in substrate 660 binding: Gln48, Gln181, Ser178, His109, His20, Asn54, Asp50, Tyr52, Glu58 661 Figure 6 Polysaccharides degradation activity of AfLPMO16. (a) CMC depolymerization: 662 estimation of reducing sugar with the increasing amount of AfLPMO16. (b) PASC 663 hydrolysis: relative absorbance at 405nm vs. AfLPMO16 quantity plot. Results are the mean 664 value of the minimum three experiments. The bar represents the standard deviation (SD) 665 Figure 7 Boosting effect of AfLPMO16. (a) Hydrolysis of alkali pre-treated rice straw: light-666 grey bar indicates only cellulase and deep-grey indicates heat inactive AfLPMO16 with 667 cellulase, dark-grey and black bar indicates cellulase along with two different quantity of 668 AfLPMO16. (b) Avicel hydrolysis: reducing sugar estimation. Light-grey bar indicates only 669 cellulase and deep-grey indicates heat inactive AfLPMO16 with cellulase, dark-grey and 670 black bar indicates cellulase along with two different quantities of AfLPMO16. (c) 671 Synergistic effect: light-grey bars indicate biomass hydrolysis by two different concentrations 672 of AfLPMO16; dark-grey bar indicating the only cellulase treated biomass and black bar 673 indicating combined treated biomass with AfLPMO16 & cellulase. Error bars represent the 674 standard deviation of experiments ran in triplicate. The different number of asterisks (*) 675 indicate a significant difference between glucose release in the presence of AfLPMO16 by 676 one-way ANOVA followed by Student's t-test (P<<0.05). 677 678 679 Enzyme Kinetics Parameter Values Vmax in U/mg 0.153 Km in mM 5.4 Kcat in min -1 277.67 680 Table 1: Enzyme kinetics of AfAA16 with 2,6, DMP as a substrate. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 23 681 682 683 684 685 686 Substrates (Biomass) Cellulases LPMOs Fold increase % increase References Wheat straw Celluclast (Novozymes) StCel61a (AA9) - 20% [46] Corn stover Celluclast (Novozymes) TaAA9 25% [50] Raw rice straw Celluclast (Novozymes) CgAA9 1.1-1.2 - [48] Raw rice straw Cellulase (MP Biomedicals) AfLPMO16 2 ~100% - Table 2: Lignocellulosic biomass hydrolysis enhancement by LPMOs (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 4, 2021. ; https://doi.org/10.1101/2020.04.24.059154doi: bioRxiv preprint https://doi.org/10.1101/2020.04.24.059154